Structural analysis of the interaction between Dishevelled2 and clathrin AP-2 adaptor, a critical step in noncanonical Wnt signaling.

Abstract

Wnt association with its receptor, Frizzled (Fz), and recruitment by the latter of an adaptor, Dishevelled (Dvl), initiates signaling through at least two distinct pathways ("canonical" and "noncanonical"). Endocytosis and compartmentalization help determine the signaling outcome. Our previous work has shown that Dvl2 links at least one Frizzled family member (Fz4) to clathrin-mediated endocytosis by interacting with the μ2 subunit of the AP-2 clathrin adaptor, through both a classical endocytic tyrosine motif and a so-called "DEP domain." We report here the crystal structure of a chimeric protein that mimics the Dvl2-μ2 complex. The DEP domain binds at one end of the elongated, C-terminal domain of μ2. This domain:domain interface shows that parts of the μ2 surface distinct from the tyrosine-motif site can help recruit specific receptors or adaptors into a clathrin coated pit. Mutation of residues at the DEP-μ2 contact or in the tyrosine motif reduce affinity of Dvl2 for μ2 and block efficient internalization of Fz4 in response to ligation by Wnt5a. The crystal structure has thus allowed us to identify the specific interaction that leads to Frizzled uptake and to downstream, noncanonical signaling events.

(A) Crystal packing in one asymmetric unit, showing the “domain swap” of the tyorine-motif segment. Each polypeptide chain is in a single color range, with the DEP domain much lighter than μ2. In the foreground are two polypeptide chains, in blue (DEP is silver-blue; μ2 is strong blue; tyrosine motif has an intermediate shade) and magenta (DEP is pink; μ2 is strong magenta; tyrosine motif is an intermediate red). These two chains swap tyrosine-motif segments, so that the blue tyrosine motif binds the receiving site on the magenta μ2 and vice-versa. (B) Domain organization of the “blue” chimera. (C) A composite structure of the DEP domain (red) and the tyrosine motif (pink) of Dvl2 bound to the C-terminal region of μ2 (μ2C) (blue).

Locations of known endocytic motifs and lipid binding site are marked. The AP-2 core structure is shown as a surface rendering (α chain in green; β2 in light blue; μ2 in blue; σ2 in yellow) and the Dvl2 DEP domain in worm representation (red).

Influence of specific mutations in μ2 on its association with the DEP domain of Dvl2

(A) Schematic representation of the Dvl2:μ2 interface; μ2C (blue) is represented as a ribbon diagram; DEP domain (pink), as a surface rendering. Residues E211, K213, K405 and F407 of μ2C, that make close contacts with the DEP domain of Dishevelled 2 are highlighted. (B) Pull-down assay with bacterially expressed wild-type or point-mutant μ2C fused to GST and His-tagged Dvl2 407–588. Bound GST-containing proteins were detected by SDS-PAGE followed by Coomassie Blue staining (50% input). Bound Dvl2 407–588 proteins were detected from a second sample processed identically, by western blot analysis with the antibody specific for the His tag in Dvl2 407–588 (50% input). The results are representative of four independent experiments. (C) Relative binding of Dvl2 407–588 to the GST-μ2C wild type and mutants used in panel B. The binding of the wild-type μ2C was set as 100%. Data correspond to average +/− std from four experiments. (D) Pull-down assay with a short peptide containing the YQRL sorting motif from TGN38 fused to GST and His-tagged Dvl2 407–588. Bound GST-containing proteins were detected by SDS-PAGE followed by Coomassie Blue staining (50% input). Bound μ2C was detected from a second sample processed identically, by western blot analysis with the antibody specific for the His tag in μ2C (50% input). The results are representative of three independent experiments.

Influence of specific mutations in the DEP domain of Dvl2 on its association with μ2 or with the AP-2 clathrin adaptor

(A) Schematic representation of the Dvl2:μ2 interface; μ2C (blue) is represented as a surface rendering and the DEP domain (pink) is shown as a ribbon diagram. Residues D441, R489 and N451 of the DEP domain at the interface with μ2C are highlighted. K446 is also shown.(B) Pull-down assay with GST-Dvl2 417–588 and AP-2 clathrin adaptor purified from calf brain coated vesicles or recombinant μ2C. Bound GST-containing proteins were detected by SDS-PAGE followed by Coomassie Blue staining (50% input). Bound AP-2 or μ2C were detected from a second sample processed identically, by western blot analysis with the antibody specific for β!2-adaptins of AP-2 or with the antibody specific for the His tag for μ2C (50% input). The results are representative of three experiments. (C) Relative binding of μ2C to the GST-Dvl2 407–588 variants used in panel B. The binding of the wild-type μ2C was set as 100%. Data correspond to average +/− std from three independent experiments.

Effect of mutations in Dvl2 that interfere with AP-2 binding on internalization of activated Fz4

(A) HEK293T cells were cotransfected with plasmids encoding HA-Fz4 (green) together with Myc-tagged wild-type mouse Dvl2 bearing the YHEL sorting motif (Dvl2 YHEL, red), Myc-tagged Dishevelled2 with AHEA instead of the YHEL motif (Dvl2 AHEA, red), Myc-tagged Dishevelled2 with the K446M mutation in the DEP domain (Dvl2 K446M, red) or Myc-tagged Dishevelled2 with the triple mutation D441K, N451A and R489A in the DEP domain (Dvl2 D441K/N451A/R489A, red). After 24 h, cells were incubated at 37°C for 10 min with an antibody specific for the HA-epitope (green), then for 20 min, also at 37°C, c with fresh medium containing Wnt5A/PMA. Cells were fixed, permeabilized and processed for immunofluorescence. Representative examples from 15 independent fields from two independent experiments for each condition are shown using equal imaging settings.(B) Analysis of images from fields corresponding to the experiments depicted in A. The extent of Fz4 internalization was scored in cells co-expressing Dvl2 and Fz4 according to previoiusly established criteria (Yu et al, 2007): (1) HA-Fz4 signal at the cell surface (none), (2) a mixture of HA-Fz4 signals at the cell surface together with a punctate intracellular pattern (partial) and (3) no discernable HA-Fz4 signal at the cell surface together with a weak intracellular punctate pattern (complete). About 300 cells were analyzed for each of the experimental conditions.